It is well-known that naturally-occurring strains of the Gram-negative microorganism Acetobacter xylinum, also known as Gluconoacetobacter xylinum (see Yamada et al., 1998, Int. J. Syst. Bacteriol. 48:327-328), are able to produce and secrete significant quantities of cellulose when grown under small-scale laboratory culture conditions wherein each microbial cell produces a single strand of cellulose commonly referred to as a strand or a fibril. Each fibril comprises multiple inter-twisted cellulose chains or microfibrils. The biochemical basis, genetics and regulation of cellulose biosynthesis in Acetobacter xylinum have been extensively studied, reported, and reviewed. Acetobacter spp. are obligate aerobic microorganisms, i.e., they have a strict requirement for O2 for respiration which drives their metabolism, growth and cellulose production. When grown in standing i.e., non-shaken/agitated, liquid cultures, Acetobacter xylinum produces pellicles comprising disorganized layers of long intertwined cellulose strands at the interfaces between the air and liquid media. As the extent and thickness of the cellulose-containing pellicle layers increase in such standing cultures, they increasingly impede then stop O2 availability from the headspace above the pellicles to the Acetobacter cells underlying the pellicles thus limiting and stopping cellulose production. Although cellulose produced by Acetobacter spp. in standing liquid cultures is chemically similar to cellulose produced from wood pulp, the major difference is that the cross-sectional diameter of Acetobacter spp. cellulose fibrils is usually about 2 orders of magnitude smaller than cellulose fibrils from wood. Typically, the cross-sectional dimensions of microfibrils produced by Acetobacter spp. is about 1.6 nm 5.8 nm, and they are twisted together to form fibrils (i.e., strands) having cross-sectional dimensions of about 3.2 nm×133 mm.
Cellulose production can be increased by culturing Acetobacter spp. in agitated liquid media wherein O2 availability to individual Acetobacter spp. cells is increased through dissolved O2 continually dispersed within and throughout the liquid media. Cellulose produced by Acetobacter spp. grown in such culture conditions is localized in multiple pellets circulating throughout the media. U.S. Pat. No. 4,863,565 and related U.S. Pat. Nos. 5,079,162, 5,144,162, 5,871,978, and 6,329,192 disclose that the macroscopic structure of cellulose in pellets produced by Acetobacter spp. cultured in agitated liquid media is characterized by a three-dimensional reticulated lattice structure that is significantly different from the layered cellulose macrostructure produced in pellicles from standing liquid cultures. The reticulated cellulose structure from liquid cultures is characterized by elongated strands of cellulose interconnected by shorter cellulosic branches or filaments having cross-section diameters of 0.1μ to 0.2μ, thereby forming grid-like patterns extending in three dimensions. The formation of the shorter cellulosic branches or filaments is apparently caused by one or more cellulose microfibrils separating out from the main fibril produced by an Acetobacter spp. cell as a result of the constant culture agitation. The shorter cellulosic branches interconnect and comingle with cellulose fibrils produced by other Acetobacter spp. cells thereby giving rise to the grid-like lattice structure. It also appears that the rates of agitation of liquid cultures significantly affect (a) the physical properties of the cellulose fibrils, strands, branches and filaments formed by Acetobacter spp., and (b) the degree of interconnecting and comingling that occurs; a low rate of agitation will result in the formation of larger cellulose-containing pellets while increasingly higher rates of agitation produce increasingly smaller cellulose pellets.
There are numerous problems encountered in attempting to scale cellulose production by Acetobacter spp. in large volumes of liquid media. For example, it appears that naturally occurring strains of cellulose-producing Acetobacter spp. are unstable when cultured in shaken or agitated liquid cultures and commonly spontaneously mutate into cellulose non-producing variants thereby limiting Acetobacter spp. cellulose production potential. As liquid culture volumes are increased, increasingly larger impellers and faster rates of impeller speeds are necessary to produce and maintain the levels of dissolved O2 required to sustain Acetobacter spp. respiration, metabolism and cellulose production. U.S. Pat. No. 4,863,565 and related U.S. Pat. Nos. 5,079,162, 5,144,162, 5,871,978, and 6,329,192, and 6,329,192 teach that shear forces in liquid media caused by high impeller speeds significantly reduce the sizes of the three-dimensional reticulated cellulose structures produced by Acetobacter spp. thereby substantially degrading the properties of the cellulose product and its commercial usefulness. Yet another problem commonly associated with cellulose production by Acetobacter spp. in both standing and agitated cultures is the propensity of these microorganisms to convert glucose to gluconic acid and/or keto-gluconic acid thereby significantly dropping the pH of the media resulting in cessation of cellulose production. Furthermore, the conversion of glucose to gluconic and keto-gluconic acids decreases glucose availability for cellulose production.
Strategies developed to address cellulose production problems associated with Acetobacter spp. include: (1) creating mutants with reduced propensity for converting glucose into acids (e.g., U.S. Pat. No. 5,079,162) or alternatively, with modified carbohydrate and/or amino acid metabolism thereby increasing rates of cellulose production (e.g., U.S. Pat. Nos. 5,962,278, 6,110,712 and 6,140,105), (2) adding cell-division inhibitors to modify and perhaps improve the physical structure and properties of cellulose produced in agitated liquid cultures (e.g., U.S. Pat. Nos. 6,060,289 and 6,627,419), (3) increasing the availability of dissolved O2 in large-volume vessels by combining two different-shaped impellers to concurrently aerate and agitate liquid media (e.g., U.S. Pat. No. 6,013,490), (4) increasing O2 availability in liquid cultures contained within vessels by increasing the amount of aeration introduced into the vessel, thereby reducing the partial pressure of CO2 while increasing the partial pressure of O2 (e.g., U.S. Pat. No. 6,017,740), and (5) post-harvest processing methods for Acetobacter spp. cellulose produced in agitated liquid cultures to improve its physical properties (e.g., U.S. Pat. No. 6,153,413). However, such strategies are complicated, costly and still have the challenge of providing sufficient O2 to enable optimal metabolism and cellulose production by Acetobacter spp. in large-volume liquid cultures.
It is well-known that other genera of obligate aerobic Gram-negative microorganisms are able to produce small amounts of cellulose from various carbon substrates under carefully controlled conditions. Such obligate aerobic cellulose-producing microorganisms include Pseudomonas sp., Alcaligenes sp., Achromobacter sp., Aerobacter sp., Azotobacter sp., Agrobacterium sp., and Rhizobium sp. isolated from sewage samples (Deinema et. al., 1971, Arch. Mikrobiol. 78:42-57), Rhizobium sp. isolated from leguminous plants (Napoli et al., 1975, Appl. Microbiol. 30:123-131), and Agrobacterium tumefaciens (Mathysse et al., 1995, J. Bacteriol. 177:1069-1075). However, the amounts of cellulose produced by these microbial genera are small relative to their carbon substrate inputs, and also, when compared to cellulose production by Acetobacter spp. Deinema et al. show in their FIGS. 1-6 on page 45 (1971, Arch. Mikrobiol. 78:42-57) that Pseudomonas sp., Aerobacter sp., Agrobacterium sp., and Azotobacter sp. produced cellulose fibrils that were branched, i.e., with microfibrils extending away from the fibrils, when grown in shaken liquid cultures. They also show in FIGS. 12 and 13 on page 48, that Pseudomonas strain (V-19-Ia) grown under the same shaken liquid culture conditions, produced elongated un-branched cellulose fibrils.
More recently, cellulose production and involvement in biofilm formation have been demonstrated in facultative anaerobic Gram negative bacteria including Escherichia coli, Klebsiella pneumoniae and Salmonella enterica (Nobles et al., 2001, Plant Physiol. 127:529-542). These species produce minute amounts of cellulose and are not expected to be of value for large-scale production.